It has long been known that acetylated histones are associated with transcriptionally active chromatin (2
). A strong correlation between histone acetylation and DNase I sensitivity has been demonstrated at the chicken β globin gene (17
). Moreover, a number of transcription factors have recently been added to the list of acetyltransferase substrates (16
). We have previously shown that the HAT CBP physically interacts with GATA-1 and stimulates its activity in an E1A-sensitive manner (4
). Interference with CBP function by forced expression of E1A led to a block in erythroid differentiation and failure to induce GATA-1-dependent genes, including the α- and β-globin genes. Deletion of the N-terminal 23 aa of E1A, which abrogates binding to CBP/p300 but not to Rb family proteins, led to loss of the ability of E1A to block GATA-1 activity and erythroid differentiation (4
). Of note, this E1A mutant retained the critical amino acids required for binding to p/CAF, which can also directly bind to E1A (34
), suggesting that the effects of E1A are largely due to specific inhibition of CBP/p300 function. Since GATA-1-binding sites in the locus control regions of the globin genes contribute to maintaining DNase I hypersensitivity, this raised the possibility that GATA-1 acts by recruiting CBP to the locus control regions, thereby locally increasing histone acetylation. The results of the present study suggest that the interaction between CBP and GATA-1 entails an additional function, namely, acetylation of GATA-1 itself.
We demonstrated that CBP acetylates murine GATA-1 in vitro at two conserved lysine-rich motifs near the two zinc fingers. Using the CBP-related protein p300, Boyes et al. (7
) mapped the major in vitro acetylation sites to the analogous motifs in chicken GATA-1, demonstrating the high degree of conservation between chicken and mouse GATA-1 in CBP- and p300-mediated acetylation.
Using anti-AK antibodies and in vivo labeling experiments we found that GATA-1 is acetylated in vivo, consistent with the findings of Boyes et al. (7
). Using site-specific GATA-1 mutants, we further showed that acetylation in intact cells occurred at the same sites acetylated by CBP in vitro.
We also demonstrate that CBP can stimulate GATA-1 acetylation in vivo at the relevant sites and that expression of E1A abolishes acetylation completely. This demonstrates that CBP can strongly stimulate the acetylation of a transcription factor in vivo and that this activity is inhibited by E1A. These data further demonstrate that GATA-1 activity correlates well with its acetylation status (4
While it is possible that other acetyltransferases such as SRC-1 and ACTR or unknown acetyltransferases acetylate GATA-1, several observations suggest that CBP is a major GATA-1 acetyltransferase. First, we have previously shown that CBP and GATA-1 associate in vivo; second, p/CAF does not acetylate GATA-1; third, there is remarkable specificity among different acetyltransferases regarding substrate and residue specificity (24
To examine the mechanism by which CBP regulates GATA-1 activity, we showed that in vitro acetylation did not detectably alter DNA binding of either full-length GATA-1 or the zinc finger region alone. Substitutions of lysine residues with arginine or alanine residues, which maintain and neutralize the positive charge, respectively, did not affect DNA-binding and dissociation rates of GATA-1 constructs expressed in mammalian cells, further supporting the lack of involvement of the acetylated motifs in DNA binding. These findings are consistent with the observation that GATA-1 mutants bearing a deletion of the entire C terminus including the C-motif (Δ308) bind DNA normally (25
). Furthermore, the solution nuclear magnetic resonance spectroscopy structure of the C-terminal zinc finger of chicken GATA-1 bound to DNA demonstrated that the site corresponding to the C-terminal acetylation motif of murine GATA-1 does not make direct contact with DNA (29
). Our findings contrast with those of Boyes et al., who reported a significant change in DNA binding upon in vitro acetylation (7
). These discrepancies might be the result of differences between chicken and mouse GATA-1 and between CBP and p300. However, in agreement with our findings, mutations in the chicken GATA-1 acetylation sites do not alter DNA binding (7
). Clearly, the strong acetylation-induced increase in DNA binding of a peptide spanning the zinc finger region of chicken GATA-1 observed by Boyes et al. in vitro is not reflected in the moderate stimulation of chicken GATA-1 activity by p300 in transient-activation assays (7
). It is possible that the in vitro conditions do not reflect the actual changes in GATA-1 function triggered upon acetylation.
Sequence analysis of in vitro-acetylated peptides revealed that lysine 312 is the major acetylated residue in the C-motif while lysine 252 and lysine 246 are the predominant sites in the N-motif. Lysine 252, which is conserved among GATA-1 of different species, residues outside the canonical RXKK motif and might account for the residual acetylation observed in vitro with the NC-mut construct. We are in the process of testing the function of GATA-1 carrying a point mutation at this site. Nevertheless, even with lysine 252 intact, mutations at the N-motif lead to reduced acetylation and diminished biological function, indicating that the N-terminal acetylation motif is important.
While sequencing of acetylated synthetic peptides allows the identification of acetylation sites in the context of an intact amino acid sequence, i.e., without the potential artifacts which might result from the introduction of mutations, the results obtained by this method must be interpreted in the context of other experimental approaches. For example, we noted that acetylation of the peptide spanning the N-motif occurred with lower efficiency than did that of the peptide spanning the C-motif. However, when assayed in the context of full-length GATA-1, disruption of the C-motif reduced acetylation to only about half of that observed with wild-type GATA-1, indicating that the extents of acetylation at the N-motif and at the C-motif are comparable. Thus, sequences outside of the stretch of amino acids contained in the peptides might contribute to maximal acetylation efficiency, perhaps by increasing the binding affinity between GATA-1 and CBP.
In performing in vitro acetylation reactions, it is important to keep in mind that recombinant, purified acetyltransferases might display a substrate spectrum distinct from that observed in the presence of additional cofactors. For example, the recombinant purified yeast acetyltransferase Gcn5 acetylates only free histones but not histones packaged into nucleosomes (8
). However, when assayed as a multicomponent complex, Gcn5 acetylates nucleosomal histones as well (for example, see reference 15
). Therefore, cofactors bound to CBP or to GATA-1 might modulate the specificity and/or efficiency of the CBP acetyltransferase.
Transient-transfection experiments designed to test the functional role of acetylation showed that the C-motif but not the N-motif of GATA-1 is required for stimulation by CBP. Since the C-motif contributes to CBP binding, this suggests that physical association or subsequent acetylation or both are important for stimulating GATA-1 activity. Mutations in the C-motif do not interfere with acetylation at the N-motif in vitro (Fig. ) or in vivo (Fig. ), suggesting that the interaction between C-mut and CBP is sufficient for efficient catalysis. To address directly whether CBP-mediated acetylation is required for GATA-1 activation, we used two CBP mutants which lack HAT activity, one containing a point mutation and the other bearing a deletion in the HAT domain. Both constructs failed to acetylate GATA-1 and histones in vitro but activated GATA-1 with an efficiency comparable to that of wild-type CBP in transiently transfected NIH 3T3 cells. Together, these findings indicate that in transient-expression assays, acetylation of GATA-1 and histones by CBP is not required for GATA-1 stimulation. At least two explanations could account for this result. First, it is important to note that in transiently transfected nonerythroid cells with artificial reporter genes, CBP might stimulate GATA-1 activity by a mechanism different from that operational in erythroid cells with fully chromatinized GATA-1 target genes. The reporter gene used in the transient-transfection assays contains a single GATA site positioned closely to a TATA box. Therefore, in this setting, it is possible that CBP augments GATA-1 activity by serving as a link between GATA-1 and the basal transcription machinery. A functional domain analysis of CBP is being used to address this question. Second, it is possible that acetylation is mediated by p300/CBP-associated acetyltransferases, including ACTR (9
) and SRC-1 (39
). Experiments are under way to test the role of the CBP-associated acetyltransferases during GATA-1 activation.
In performing similar transient-transfection experiments, Boyes et al. observed that a p300 mutant bearing a 50-aa deletion in the HAT domain failed to stimulate chicken GATA-1 activity (7
). It is possible that such a deletion abrogated functions of p300 unrelated to its HAT activity.
While Boyes et al. did not test the function of the C-motif (the strongest acetylation site), mutations in the chicken GATA-1 N-motif resulted in a reduced response to p300, leading the authors to establish a correlation between chicken GATA-1 acetylation at the N-motif and transcriptional activation. Two open questions remain regarding this interpretation: first, the observed reduction in the p300 response is marginal (2.5-fold in wild-type GATA-1 compared to 1.4- and 1.9-fold in two acetylation mutants), especially in a transient-transfection assay. Second, the authors did not provide information about the acetylation status of these sites in vivo. In our experiments, mutations in the N-motif had no effect on DNA binding or transactivation by CBP in transient-transfection assays. Furthermore, binding of GATA-1 to a single GATA-1 site does not require the N-terminal zinc finger (25
). Therefore, we believe that the mechanism by which the N-motif contributes to the GATA-1 function remains to be elucidated but is unlikely to involve regulation of DNA binding. In evaluating these findings, we point out that a biological function of the N-motif required the assay of GATA-1 constructs in erythroid cells with GATA-1-dependent genes present in their natural state (see below).
Since critical GATA-1 functions were uncovered only in maturing erythroid cells (6
), we used the GATA-1-deficient cell line G1E to study the functional role of the acetylation sites. Mutations in either the N- or C-motif significantly reduced activity, while mutations in both motifs abrogated activity entirely. Thus, only in erythroid cells did we uncover a requirement for the N-motif. Mutations in the N-motif did not interfere with binding to CBP or to Fog (data not shown), the recently identified cofactor which selectively binds to the N-terminal zinc finger of GATA-1 (41
), excluding the possibility that mutations simply disrupt the interaction of GATA-1 with its known cofactors. Deletion of the C terminus of GATA-1 leaving the C-motif intact (Δ331) resulted only in a small loss of activity, whereas a slightly larger deletion which removes the C-motif (Δ308) led to a much more pronounced loss of function without affecting DNA binding. Together, these results demonstrate that mutations in the acetylation motifs impair biological activity without affecting DNA binding, which demonstrates that the DNA-binding and acetylation functions can be uncoupled.
Interestingly, the ability of GATA-1 to induce megakaryocytic conversion of the early myeloid cell line 416B also depends in part on an intact C-motif (42
). In these assays, Δ308 was significantly less active than Δ331, which raises the possibility that CBP also synergizes with GATA-1 in the regulation of megakaryocytic gene expression. Together, these results indicate that the C-terminal acetylation site is of great biological importance and are consistent with a requirement of CBP for GATA-1 function in vivo.
Acetylation of lysine residues neutralizes their positive charge and changes the size of the residue. In general, such changes could conceivably entail alterations in protein function similar to those incurred upon phosphorylation. For example, acetylation could lead to changes in protein conformation. Such a mechanism has been proposed for p53, where acetylation in the regulatory domain leads to increased DNA-binding activity (16
). Alternatively, acetylation could directly influence protein-DNA interactions, as has been suggested for binding of histone tails to DNA, or it could affect protein-protein interactions, as described for binding of histone tails to the yeast transcriptional repressor Tup1 (13
The mechanism by which either acetylation motif in GATA-1 exerts its function in G1E cells remains to be determined. Gel shift analysis with nuclear extracts of the virally infected G1E cells demonstrated the presence of equal amounts of DNA-bound GATA-1 proteins, and immunofluorescence microscopy confirmed the nuclear localization of all constructs. This suggests that acetylation in either motif does not regulate nuclear localization, DNA binding, or protein stability. Several additional mechanisms by which acetylation regulates GATA-1 activity could be envisioned. It is conceivable that acetylation leads to a conformational change in GATA-1, resulting in increased activity through exposure of an activation domain. Alternatively, the acetylation motifs might represent docking sites for still unidentified coactivators or repressors, and acetylation might positively or negatively regulate their binding affinity. More extensive studies, including structural analyses, will be required to determine the mechanistic role of GATA-1 acetylation.